-
Journal of Surface Engineered Materials and Advanced Technology,
2013, 3, 269-274 http://dx.doi.org/10.4236/jsemat.2013.34036
Published Online October 2013
(http://www.scirp.org/journal/jsemat)
Adsorption of α-Chymotrypsin on Plant Biomass Charcoal
Hidetaka Noritomi1*, Keito Hishinuma1, Shunichi Kurihara1,
Jumpei Nishigami1, Tetsuya Takemoto2, Nobuyuki Endo3, Satoru
Kato1
1Department of Applied Chemistry, Tokyo Metropolitan University,
Minami-Ohsawa, Hachioji-shi, Tokyo, Japan; 2Osaka Gas Co., Ltd.,
Konohana-ku, Osaka, Japan; 3EEN Co., Ltd., Bunkyo-ku, Tokyo, Japan.
Email: *[email protected] Received June 11th, 2013; revised July
13th, 2013; accepted August 2nd, 2013 Copyright © 2013 Hidetaka
Noritomi et al. This is an open access article distributed under
the Creative Commons Attribution Li-cense, which permits
unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.
ABSTRACT The adsorption of α-chymotrypsin onto plant biomass
charcoal (PBC), which was prepared from plant biomass wastes such
as bagasse and dumped adzuki beans by pyrolysis, has been examined.
The PBC was characterized by SEM, spe-cific surface area, and pore
size distribution. The adsorption isotherms were successfully
correlated by the Freundlich equation. The amount of α-chymotrypsin
adsorbed on PBC was dramatically dependent upon the solution pH and
tem-perature. Maximum adsorptions of α-chymotrypsin on adzuki bean
charcoal and bagasse charcoal were observed at weak acidic and near
neutral pH, respectively. The amount of α-chymotrypsin adsorbed on
PBC decreased with an in-crease in the concentration of salts.
Plots of the amount of α-chymotrypsin adsorbed on PBC versus
temperature exhib-ited an optimum temperature. Keywords:
Adsorption; Characterization; Plant Biomass Charcoal;
α-Chymotrypsin; Protein
1. Introduction The use and application of boimass for renewable
re-sources and energies are one of the most important chal-lenges
to establish a recycling society. Recently, much attention has been
given to the charcoal prepared from plant biomass wastes in order
to use soil amendments, adsorbents, humidity control materials,
materials for wastewater treatment, and catalysts [1-6]. However,
plant biomass wastes have not sufficiently been recycled yet,
compared to other wastes, although an enormous amount of plant
biomass waste has been discharged in the world. Moreover, the
development in the high value-added func- tion of charcoal derived
from plant biomass wastes has been desired.
The adsorption of proteins onto the surface of solids has been
studied extensively in the biotechnological, medical,
pharmaceutical, and food fields in order to ap-ply it to the
immobilization of biocatalyst in the bioreac-tor, the separation of
proteins, and the removal of protein contamination from food and
medicine [7,8]. In order to assess the property of plant biomass
charcoal (PBC) as a biomaterial, we have so far investigated the
interaction between a protein and PBC derived from dumped
adzuki
beans and so on, when hen egg white lysozyme (HEWL) was used as
a model protein, and have found out that PBC effectively adsorbs
HEWL, and HEWL adsorbed on PBC exhibits the enhanced storage
stability at low tem- peratures and the excellent thermal stability
at high tem- peratures, compared to those of native HEWL
[9-11].
In the present work, to test the generality on the ad-sorption
efficiency of PBC for proteins, we employed bovine pancreas
α-chymotrypsin as a model protein, since it is well investigated
regarding its structure, functions, and properties [12]. In
addition to adzuki bean charcoal and bamboo charcoal, which were
used in our previous work [9], bagasse charcoal and wood charcoal
have new- ly been used as PBC. Moreover, we investigated structu-
ral characteristics of PBC.
2. Experimental 2.1. Materials
α-Chymotrypsin (EC 3.4.21.1 from bovine pancreas) (type II, 52
units/mg solid) was purchased from Sigma- Aldrich Co. Plant biomass
charcoal derived from ba-gasses was supplied from Osaka Gas Co.
Ltd. Plant bio-mass charcoals derived from adzuki beans, bamboos,
and woods were from EEN Co. Ltd. Medicinal carbon was
*Corresponding author.
Copyright © 2013 SciRes. JSEMAT
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Adsorption of α-Chymotrypsin on Plant Biomass Charcoal 270
100 nm
B
100 nm
CA
100 nm
100 nm
E
100 nm
D
Figure 1. SEM images of (A) bagasse charcoal, (B) adzuki bean
charcoal, (C) bamboo charcoal, (D) wood charcoal, and (E) medicinal
carbon. obtained from Nichi-Iko Pharmaceutical Co. Ltd.
2.2. Characterization of Biomass Charcoal Powder
The SEM micrograph was obtained using a scanning electron
microscope (JSM-7500F FE-SEM, JEOL Ltd.) operating at 5 or 15 kV.
The sample for SEM was pre- pared on a carbon tape without vapor
deposition.
All samples were outgassed at 300˚C for 8 h prior to the
nitrogen adsorption measurements. The specific sur-face area of PBC
was calculated with the use of the Brunauer-Emmett-Teller (BET)
method using a micro-pore system (BELSORP-mini II, BEL JAPAN,
INC.).
2.3. Adsorption of α-Chymotrypsin onto Plant Biomass
Charcoal
As a typical procedure, 5 mL of 0.01 M phosphate buffer solution
at pH 7 containing 300 μM α-chymotrypsin and 3 g/L bagasse charcoal
was placed in a 10-mL test tube with a screw cup, and was incubated
at 25˚C and 120 rpm for 24 h. After adsorption, the mixture was
filtrated with a membrane filter (pore size: 0.1 μm, Millipore Co.
Ltd.). The amount of α-chymotrypsin adsorbed on PBC was calculated
by subtracting the amount of α-chymo- trypsin included in the
supernatant liquid after adsorption from the amount of
α-chymotrypsin in the aqueous solu- tion before adsorption. The
amount of α-chymotrypsin was measured at 280 nm by UV/vis
spectrophotometer (UV-1800, Shimadzu Co. Ltd.).
The aqueous solutions used in this study were acetate buffer
solutions at pH 4 and 5, phosphate buffer solutions at pH 6 and 7,
borate buffer solutions at pH 8, 9, and 10, and disodium hydrogen
phosphate buffer solutions at pH 11 and 12. The concentration of
buffer solution was pre-pared at 0.01 M. Each data point for the
amount of α- chymotrypsin adsorbed represents an average of three
measurements with a standard error less than 10%.
3. Results and Discussion 3.1. Characterization of Plant Biomass
Charcoal In order to confirm the morphology of PBC, we have
obtained SEM images presented in Figure 1 for bagasse charcoal,
adzuki bean charcoal, bamboo charcoal, and wood charcoal.
Additionally, SEM image of medicinal carbon is shown as typical
activated carbon in Figure 1. As seen in the figure, the morphology
of PBC surface is strongly dependent upon the kind of materials.
Bagasse charcoal was produced under 600˚C by a charcoal kiln.
Adzuki bean charcoal, bamboo charcoal, and wood char coal were
prepared under 450˚C by pyrolysis without combustion under a
nitrogen atmosphere [9]. Conse- quently, the preparation of PBC
used in the present work was not carried out by activation
treatment. On the other hand, as seen in Figure 1(E), the surface
of medicinal carbon was obviously much rougher than that of PBC,
and many pores were observed on the surface.
Table 1 shows the textural parameters of PBC ob-tained from
low-temperature (−196˚C) nitrogen adsorption
Copyright © 2013 SciRes. JSEMAT
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Adsorption of α-Chymotrypsin on Plant Biomass Charcoal 271
Table 1. Structural characteristics of PBC.
Charcoals Specific surface area [m2/g] Pore volume [cm3/g] Pore
diameter peak [nm]
Bagasse 459 0.047 less than 2.6
Adzuki bean 204a - -
Bamboo 294 0.041 less than 2.6
Wood 117 0.025 less than 2.6
Medicinal carbon 1158 0.32 less than 2.6
aSpecific area of adzuki bean charcoal was obtained from the CO2
isotherm. isotherms, which allow the calculation of specific
surface area, specific pore volume, and pore diameter peak. In the
table, the specific area of adzuki bean charcoal de-picted the
value obtained from the CO2 isotherm in our previous work [9],
since it was too long to reach the ad-sorption equilibrium, and the
exact value could not be obtained. The specific surface area of PBC
was much smaller than that of medicinal carbon, and the specific
pore volume of PBC was one order of magnitude lower than that of
medicinal carbon. The characteristics of pores and surface
chemistry of charcoal are influenced by carbonizing temperature
[13]. The specific pore vol-ume tends to increase as the
carbonizing temperature of charcoal increase. It was presumed that
the formation of pores of PBC was not enhanced, since PBC was
prepared at low temperatures.
Figure 2 shows the pore size distribution of PBC ob- tained by
the Barrett-Joyner-Halenda (BJH) method [14], which is based on a
model of the adsorbent as a collec- tion of cylindrical pores. The
theory accounts for capil- lary condensation in the pores using the
classical Kelvin equation, which in turn assumes a hemispherical
liquid- vapor meniscus and a well-defined surface tension. The pore
size distribution of PBC was less than 10 nm, while that of
medicinal carbon was less than 100 nm. Thus, the pore size of PBC
was mainly in the micro-pore range, whereas that of medicinal
carbon was in the meso-pore and macro-pore ranges.
3.2. Adsorption Isotherms We used α-chymotrypsin as a model
protein. The amount of α-chymotrypsin adsorbed on BCP increased
with an increase in the time of adsorption, and reached a plateau
around 24 h. Figure 3 shows the amount of α-chymo- trypsin adsorbed
on PBC. The amount of α-chymotrypsin adsorbed varied with the kind
of materials. As seen in Figure 3, the sequence of the amount
adsorbed went as follows: medicinal carbon > bagasse charcoal
> wood charcoal > adzuki bean charcoal > bamboo charcoal,
while that of the specific surface area went as follows: medicinal
carbon > bagasse charcoal > bamboo charcoal > adzuki bean
charcoal > wood charcoal, as shown
0
0.01
0.02
0.03
0.04
0.05
0.06
1 10 100 1000
Bagasse charcoalBamboo charcoalWood charcoalMedicinal charcoal
dp (nm)
dV
p/dd p
Figure 2. Pore size distribution of plant biomass charcoals.
0 10 20 30 40 5
Medicinal carbon
Wood charcoal
Bamboocharcoal
Adzuki bean charcoal
Bagasse charcoal
Amount adsorbed (µmol/g)
0
Figure. 3 Effect of the kind of materials on the amount of
α-chymotrypsin adsorbed; adsorption was carried out by incubating
buffer solution (pH 7) containing 300 μM α- chymotrypsin and 3 g/L
PBC or medicinal carbon at 120 rpm and 25˚C for 24 h. in Table 1.
Concerning PBC, the amount adsorbed did not correspond to the
specific surface area. As seen in Figures 1 and 2, the morphology
of PBC surface and the pore size distribution of PBC markedly
depended upon the kind of raw materials. Consequently, it is
assumed that they affect the adsorption efficiency of PBC. The
Copyright © 2013 SciRes. JSEMAT
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Adsorption of α-Chymotrypsin on Plant Biomass Charcoal 272
amount of α-chymotrypsin on bagasse charcoal was 0.6- fold that
on medicinal carbon, although the surface area of medicinal carbon
was about 2.5 times larger than that of bagasse charcoal. The scale
of α-chymotrypsin is 5.1 × 4.0 × 4.0 nm [12]. Thus, it is
considered that pores hav- ing less than the size of proteins do
not work effectively against the adsorption of proteins. This
indicates that ba- gasse charcoal has reasonable adsorption
efficiency for α-chymotrypsin.
Figure 4 shows the adsorption isotherms of α-chy- motrypsin on
adzuki bean charcoal and bagasse charcoal. These isotherms exhibit
a gradual increase. The amount of α-chymotrypsin adsorbed on
bagasse charcoal is supe- rior to that of α-chymotrypsin adsorbed
on adzuki bean charcoal. The solid lines presented in the figure
are the best fit Freundlich isotherm characterization of the ex-
perimental data using Equation (1).
1/F
nW K C (1)
Here, W is the amount of α-chymotrypsin adsorbed on PBC, C is
the α-chymotrypsin concentration, and KF and n are experimental
constants [15]. The correlation con- stants (r2) of adzuki bean
charcoal and bagasse charcoal were both 0.990. With regard to other
adsorption iso- therm models, for example, when the data were
fitted for the adsorption isotherm model of Langmuir, the correla-
tion constants (r2) of adzuki bean charcoal and bagasse charcoal
were 0.853 and 0.815, respectively. The present isotherm type was
similar to that of the adsorption of HEWL onto PBC, and
α-chymotrypsin was more effec- tively adsorbed on PBC, compared
with HEWL [9]. The isotherm of bagasse charcoal displayed upward
curvature, compared to that of adzuki bean charcoal. This
indicates
0
5
10
15
20
25
30
0 100 200 300 400
Amou
nt a
dsorb
ed(μm
ol/g
)
F inal solution concentration(μM)
Adzuki bean charcoalBagasse charcoal
Figure 4. Adsorption isotherms of α-chymotrypsin on ad-zuki bean
charcoal and bagasse charcoal; adsorption was carried out by
incubating buffer solution (pH 7) containing a certain amount of
α-chymotrypsin and 3 g/L PBC at 120 rpm and 25˚C for 24 h.
that the adsorption of α-chymotrypsin on bagasse char- coal is
more effective than that on adzuki bean charcoal.
3.3. Effect of pH Value on α-Chymotrypsin Adsorption
Figure 5 shows the relationship between the pH value of aqueous
solutions and the amount of α-chymotrypsin adsorbed on adzuki bean
charcoal and bagasse charcoal at 25˚C. The amount of α-chymotrypsin
adsorbed on ba- gasse charcoal sharply increased with an increase
in the pH value, reaching the optimum value around neutral pH, and
tended to decrease in the alkaline region. The pH profile in the
case of adzuki bean charcoal was similar to that in the case of
bagasse charcoal, although the optimal value of adzuki bean
charcoal is slightly shifted to acidic pH, compared to that of
bagasse charcoal. The pH profile in the adsorption of
α-chymotrypsin on PBC was similar to that in the adsorption of HEWL
on PBC [9]. The net charge on the protein molecules is varied by
adjusting the pH of the solution, since the protein molecule is
con- structed by amino acid residues containing positive- and
negative-charged side chains. α-Chymotrypsin belongs to basic
proteins, and the isoelectric point (pI) of α-chymo- trypsin is 9.1
[12]. The lower the pH of solution contai- ning α-chymotrypsin
becomes below the pI of α-chymo- trypsin, the more positive the net
charge of α-chymo- trypsin becomes. The ζ potential of PBC
drastically de-creases with increasing the pH value, exhibiting a
nega- tive value above pH 4, drops till pH 7, and is almost con-
stant in the alkaline region [9]. When the pH value was around the
pI of α-chymotrypsin or the pH where the ζ
0
5
10
15
20
25
0 2 4 6 8
Am
ount
ads
orb
ed(
μm
ol/
g)
pH [-]
10
Adzuki bean charcoal
Bagasse charcoal
Figure 5. Effect of pH on the amount of α-chymotrypsin ad-
sorbed on adzuki bean charcoal and bagasse charcoal; ad- sorption
was carried out by incubating buffer solution (ap- propriate pH)
containing 300 μM α-chymotrypsin and 3 g/L PBC at 120 rpm and 25˚C
for 24 h.
Copyright © 2013 SciRes. JSEMAT
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Adsorption of α-Chymotrypsin on Plant Biomass Charcoal 273
potential of PBC approached 0 volts, a dramatic decrease in the
amount adsorbed was observed. The electrostatic interaction between
the positive charge of α-chymotryp- sin and the negative charge on
PBC tends to decrease in the region of acidic or alkaline pH.
Therefore, in the vi- cinity of neutral pH where the Coulomb force
between PBC and α-chymotrypsin is high, a high amount of ad-
sorption tends to be obtained. Consequently, the adsorp- tion
profiles seem to be related mainly with the electro- static
interaction.
3.4. Effect of Ionic Strength on α-Chymotrypsin Adsorption
Figure 6 shows the relationship between the KCl con- centration
of the solution and the amount of α-chy- motrypsin adsorbed on
adzuki bean charcoal and bagasse charcoal. The amount of
α-chymotrypsin adsorbed on PBC decreased with an increase in KCl
concentration. The solutions of α-chymotrypsin at the KCl
concentra- tion employed at the present work were transparent, and
no precipitate was observed. At first, it is considered that an
increase in the ionic strength results in a decrease in the
electrostatic interaction due to the electrostatic screen- ing
effect. Many radical species due to functional groups containing
oxygen atoms, which are formed by thermal decomposition of
cellulose and hemicelluloses, are de- tected in charcoals
carbonized at 500˚C by the measure- ment of electron spin
resonance, and functional groups decrease with increasing
carbonization temperature [13, 16]. We have reported that the
elemental ratio of oxygen on the surface of PBC was more than 15%,
and C-O,
0
5
10
15
20
25
30
0 0.001 0.01 0.1
Am
ount
adso
rbed (
μm
ol/
g)
KCl concentration(M)
Adzuki bean charcoal
Bagasse charcoal
Figure 6. Effect of KCl concentration on the amount of α-
chymotrypsin adsorbed on adzuki bean charcoal and ba- gasse
charcoal; adsorption was carried out by incubating buffer solution
(pH 7) containing 300 μM α-chymotrypsin, 3 g/L PBC, and a certain
amount of KCl at 120 rpm and 25˚C for 24 h.
O-C-O, C=O, and COOH were detected by X-ray photo- electron
spectroscopy [9]. Consequently, it is suggested that electrostatic
interactions and hydrogen bondings via functional groups on the
surface of PBC largely contrib- ute to the adsorption of
α-chymotrypsin, since α-chy- motrypsin has many ionic and hydroxyl
amino acid resi- dues, as seen in Figure 7. Thus, the addition of
inorganic salts appears to weaken those interactions between PBC
and α-chymotrypsin.
3.5. Effect of Temperature on α-Chymotrypsin Adsorption
Figure 8 shows the plots of the amount of α-chymotryp- sin
adsorbed on adzuki bean charcoal and bagasse char- coal against the
adsorption temperature. The amount of α-chymotrypsin adsorbed on
PBC was dramatically in- fluenced by the temperature. The maximum
amounts of α-chymotrypsin adsorbed on adzuki bean charcoal and
bagasse charcoal were both observed around 25˚C. This tendency was
similar to the case of the adsorption of HEWL onto PBC [9]. The
temperature profile on the amount of proteins adsorbed on the
water-insoluble ma- trix tends to exhibit an optimum temperature,
since the conformation of proteins is generally sensitive to tem-
perature [17]. The state on the surface of protein molecules
Plant Biomass CharcoalPlant Biomass Charcoal
Functional Group
Protein
Figure 7. Schematic representation of adsorption of pro-teins
onto the surface of PBC.
0
5
10
15
20
25
30
0 10 20 30 40 5
Am
oun
t ads
orbe
d (μ
mol
/g)
Temperature(℃)
0
Adzuki bean charcoal
Bagasse charcoal
Figure 8. Effect of temperature on the amount of α-chy-
motrypsin adsorbed on adzuki bean charcoal and bagasse charcoal;
adsorption was carried out by incubating buffer solution (pH 7)
containing 300 μM α-chymotrypsin and 3 g/L PBC at 120 rpm and an
appropriate temperature for 24 h.
Copyright © 2013 SciRes. JSEMAT
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Adsorption of α-Chymotrypsin on Plant Biomass Charcoal
Copyright © 2013 SciRes. JSEMAT
274
such as the charge, hydrophilicity, and hydrophobicity due to
their conformation affects the interaction of pro- teins with
matrices. Moreover, the sufficient potential to weaken the
hydration layer around a protein molecule is necessary to enhance
the interactions between the amino acid residues of proteins and
functional groups on the surface of PBC. Therefore, since the
entropy effect of protein and the enthalpy effect of adsorption
phenome- non are involved in the adsorption, the adsorption profile
tends to exhibit the maximum at an appropriate tempera- ture.
4. Conclusion PBC had the adsorption efficiency for proteins,
similar to medicinal carbon. The adsorption isotherms followed the
Freundlish equation. PBC exhibited the optimum pH on the amount
adsorbed due to the interaction between α- chymotrypsin and PBC,
such as the electrostatic force, the hydrogen bonding. The
adsorption temperature mar- kedly affected the amount adsorbed.
5. Acknowledgements This work was supported by a Grant-in-Aid
for Scientific Research (C) from Japan Society for the Promotion of
Science (No. 24561013).
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